In order to detect and discriminate numerous odorants encountered in the environment, the mammalian olfactory system employs a large family (>1000 in rodents) of odorant receptors (ORs), which belong to the superfamily of seven transmembrane (TM), G protein-coupled receptors (GPCRs). The olfactory epithelium in the nasal cavity harbors a few million olfactory sensory neurons (OSNs) with each neuron expressing a single OR type. Despite the diversity of OR proteins, they are all coupled to common G proteins which trigger a well- characterized cAMP transduction cascade to transform the chemical signals into electrical signals. However, little is known about the molecular and structural mechanisms underlying receptor-ligand binding and G protein activation. The consensus model in the field is that ORs are used combinatorially to encode odor identities with each OR capable of interacting with a small number of specific ligands. We recently discovered a mouse OR (SR1 or MOR256-3) with unconventional properties; i.e., it responds to many odorants with diverse size, shape, and functional groups. In addition to broad responsiveness, genetically labeled SR1 neurons also show mechanical responses to pressure changes, which are likely mediated by a similar cAMP cascade for odorant responses. Using SR1 and a few classical, selective ORs as models, the long-term goal of this project is to understand the molecular and structural basis for OR tuning properties and OR-G protein interactions. By combining patch-clamp, gene-targeting, site-directed mutagenesis, and heterologous expression approaches, we will specifically test the following hypotheses. First, the broadly-tuned SR1 has a lower activation threshold so that SR1 neurons have a higher spontaneous activity level than those expressing narrowly-tuned ORs. Furthermore, amino acid variations in two highly-conserved domains near the cytoplasmic ends of TM3 and TM6 cause gain-of-function phenotype in broadly-tuned ORs such as SR1, which has uncharacteristic sequences in these regions. Consequently, even non-preferred ligands can cause sufficient conformational changes in the receptor to activate G proteins. Second, broadly-tuned ORs serve as mechanical sensors in the host OSNs and mechanical stimulation (such as that carried by the airflow) can induce enough conformational alterations to activate the receptor and subsequently the G protein cascade. Third, various G proteins play different roles in shaping the selectivity and sensitivity of OSNs. Some narrowly-tuned ORs can confer broad responsiveness and mechanosensitivity to OSNs under more permissive cellular conditions. Overall, carrying out these studies will provide new insights into the molecular basis for the tuning properties of ORs and the cellular features defining the specificity and mechanosensitivity of OSNs. This project will advance our knowledge on how odor and airflow information is encoded and processed by the olfactory system. PUBLIC HEALTH RELEVANCE: Millions of people, especially the elderly, suffer from smell dysfunction, which poses a negative impact on their quality of life. A better understanding on the molecular and structural basis for ligand recognition by odorant receptors will help to develop medical treatments to enhance the desired smell functions. These studies will also help to design better therapeutic agents targeting other G protein-coupled receptors, which play pivotal roles in signal transduction in all body organs.